1. Field of the Invention
The present invention relates generally to genetic compositions and recombinant methodology from the preparation of Trichomonas vaginalis proteins and peptides, useful, e.g., in the detection, identification and characterization of Trichomonas vaginalis organisms, as well as in the development of vaccines.
2. Description of the Related Art
Trichomonas vaginalis remains one of the most poorly investigated infectious agents; yet it is the most prominent parasite causing an illness in developed countries. In all world societies trichomoniasis causes an economic and emotional burden equal to other devastating pathogens of microbial etiology.
Trichomoniasis is a non-self-limiting, gender-discriminating disease of women. Infection of men with T. vaginalis will mostly be asymptomatic and self-limiting. A major problem with this disease is the broad spectrum of symptomatology, ranging from asymptomatic carriers who are reservoirs for the parasite to patients with a foul-smelling bloody discharge, severe inflammation and discomfort. For reasons not known at present, asymptomatic women can become highly symptomatic, and host or parasite factors which help explain the susceptibility or resistance to T. vaginalis infection and/or trichomoniasis remain undefined.
The Trichomonas vaginalis parasite is a flagellated protozoan responsible for a world-wide sexually transmitted disease (Ackers, 1982; Honigberg, 1978; Krieger, 1981; Muller, 1983; Rein and Chapel, 1975). Yearly estimates of numbers of patients with trichomoniasis in the United States alone range from 4 million to as high as 10 million. Unfortunately, it is estimated that fifty percent of all patients will go undiagnosed using the standard procedure of visualization of wet-mount preparations (Spence et al., 1980). Alternative diagnostic methods, such as culturing of the parasite from vaginal swabs, are expensive, time consuming, and not readily available.
The differentiation among trichomonal isolates, based on pathogenicity levels inherent to individual isolates, have not been satisfactorily established (Krieger et al., 1990), even using rodent animal models (Honigberg et al., 1966) which do not mirror the human infection. This absence of an animal model remains a major impediment toward studying the extent and nature of sequelae caused by T. vaginalis parasitism. Recently, epidemiologic evidence points toward a possible predisposition of certain women with trichomoniasis for HIV infection (Laga et al., 1989; Laga et al., 1990). Finally, although nitroimidazoles used to treat trichomoniasis in humans are extremely efficacious, the drugs have toxic side-effects, and drug refractoriness by trichomonal isolates has been documented (Muller et al., 1980).
Independent investigations have demonstrated a dramatic variation in the antigenic structure of T. vaginalis isolated form patients (Krieger et al., 1985; Su-Lin et al., 1983; Teras, 1960). It has more recently been appreciated that the surface disposition of a repertoire of high molecular weight prominent immunogens may be responsible for some of this antigenic diversity (Alderete et al., 1985b; Alderete et al., 1986a; Alderete et al., 1987a). Through the application of immunological techniques, sera from patients with trichomoniasis have been classified as either Type I or Type II, depending on the presence or absence of various immunologic reactivities (Alderete et al., 1987b). For example, differential receptivity with an immunogen having a M.sub.r of .about.270 (P270) is one means of distinguishing Type I and Type II isolates, with Type I isolates being a homogeneous population lacking P270 expression or their surface (Alderete et al., 1986c; Alderete et al., 1986d). So-called Type II isolates generally comprise a heterogeneous population with regard to surface expression of the P270 immunogen (Alderete 1987b).
Indirect immunofluorescence using the pooled patient sera demonstrated the heterogeneous nature of parental trichomonal populations (Alderete 1985b). Those isolates, which gave strong reactions by the whole cell-radioimmunoprecipitation assay (Alderete 1983b) mentioned above, were comprised of subpopulations of parasites in which some were fluorescent and some were non-fluorescent with the patient sera (Alderete et al., 1985b; Alderete 1987a). This earlier work also revealed the fascinating aspect of changing proportions of fluorescent and non-fluorescent parasites during in vitro cultivation, and this now required clarification.
The generation of monoclonal antibodies (MAbs) reactive to T. vaginalis surfaces was fortuitous, since one MAb (C20A3) gave cytofluorometric patterns among the various trichomonal isolates identical to those detected with the pooled sera from patients (Alderete et al., 1985b; 1986d; and 1987a). This MAb was a key reagent for confirming the property of protein phenotypic variation based on the surface expression of immunogens (Alderete et al., 1986c and 1987b), and the existence of two types of parental populations of T. vaginalis parasitizing humans was further demonstrated (Alderete et al., 1986c; 1986d; Alderete et al., 1987b).
Type I isolates are trichomonads that synthesize but do not undergo phenotypic variation for the surface expression of the immunogens. At the present time approximately 40% of women appear infected with Type I isolates. Type II isolates, then, are trichomonads capable of phenotypic variation. It is noteworthy that only 1/3 of women with Type II isolates harbor both fluorescent and non-fluorescent trichomonads in the vagina, but the numbers of fluorescent trichomonads are small in comparison to MAb non-fluorescent parasites (Alderete et al., 1987b). In other words, the in vivo environment apparently favors or selects for T. vaginalis organisms lacking the expression of the surface immunogen repertoire although upon in vitro cultivation the parasites readily revert to the opposite phenotype. This property was also in part experimentally demonstrated by showing the isolation of only non-fluorescent trichomonads after subcutaneous inoculation of mice hindquarters with only MAb C20A3-fluorescent parasites (Alderete 1987a). Nonetheless, the fact that 90% of women make antibody to the C20A3-reactive immunogen (Alderete et al., 1987b) showed the molecule was synthesized regardless of the Type or subpopulation designations of the infecting T. vaginalis isolate.
Of further interest is the delineation of three subpopulations of parasites among the Type II isolates (Alderete et al., 1986c). For example, in addition to the non-fluorescent organisms some, but not all, fluorescent trichomonads were readily killed in a complement-independent fashion after exposure to the C20A3 MAb (Alderete, 1986b). The lysis of some parasites expressing the C20A3-reactive immunogen might indicate an important biofunctionality for the molecule; however, T. vaginalis without surface immunogen are equally capable of in vitro and in vivo growth and multiplication.
The complexity of the surface immunogens with regard to antibody accessibility was further reinforced in these studies by the identification of another high molecular weight protein by a MAb called DM126. The protein was found on the surface of parasites of all isolates (Alderete et al., 1987b); however, flow cytofluorometry with DM126 again indicated the existence of homogeneous, non-fluorescent and heterogeneous populations as seen for the MAb C20A3. The accessibility of the immunogen to antibody binding was highly variable in spite of its presence on the trichomonal surface, indicating that conformational changes were occurring for this protein possibly depending upon the immediate microenvironment of the parasite. This property was termed epitope phenotypic variation.
More recently, the availability of monospecific antiserum to the purified immunogen has revealed the predominance of this molecule throughout trichomonal surfaces. Of particular interest, however, was the refractoriness of the parasites to complement-mediated killing in the presence of this monospecific antiserum. Although a precise chemical-molecular explanation for this property is unknown, it illustrates the overall resistance of the protozoan to killing by antibody against common, stable immunogens which might otherwise be targets for vaccine development strategies. These immune evasion capabilities certainly will allow the parasite to survive in the face of host antibody responses.
Curiously, Type II isolates predominate in vivo, a finding which suggests that Type II isolates may be more virulent. If this is the case, P270 may be an excellent molecular marker for virulent isolates. Importantly, the P270 immunogen elicits the highest levels of antibody in experimentally infected animals (Alderete et al., 1986d), and most, if not all, trichomoniasis patients make antibody to this molecule (Alderete et al., 1987b). The significance of the highly immunogenic nature of P270 and its role in host-parasite biology may prove important to an overall understanding of this major pathogen.
Other than the discovery of the P270 immunogen, little is known regarding the biology of T. vaginalis on a molecular level. Furthermore, very few molecular tools are presently available to the researcher or clinician which will allow the identification and characterization of T. vaginalis isolates. In particular, there are currently no reported methods for obtaining recombinant DNA agents for the direct identification and characterization of T. vaginalis isolates, or for use in producing T. vaginalis proteins or peptides, particularly antigenic or immunogenic proteins or peptides. Accordingly, there is currently a great need for recombinant methodology and genetic constructs which are applicable to T. vaginalis, particularly methodology directed to the identification of virulent isolates, or distinguishing between Type I and Type II isolates.